This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

As a known regulator of apoptosis, survivin has positive relationship with lymphatic
metastasis in breast cancer. This study aims to detect the difference in expression
between survivin and vascular endothelial growth factor-C (VEGF-C) in treated breast
cancer cells and tissues, and to analyze the correlation among survivin, VEGF-C and
lymphatic metastasis.

Methods

Plasmid with survivin and VEGF-C shRNA and lentivirus with survivin gene were constructed
and transfected into breast cancer cell ZR-75-30. Then the expressions of the two
genes were examined using western blot analysis and real-time PCR. The change of invasiveness
of breast cancer cells was assessed using matrigel invasion assay. Using immunohistochemistry,
the expression of survivin and VEGF-C were analyzed in 108 clinical breast cancer
cases with breast cancer tissue and lymph node.

Results

Survivin regulated the expression of VEGF-C at both protein and mRNA levels in breast
cancer cells. Immunohistochemical analysis showed that the level of VEGF-C expression
was significantly related with that of survivin in breast cancer tissues (p<0.05). VEGF-C was found to participate in the process of breast cancer cells invasion
mediated by survivin. The co-expression of the two and the single expression of any
one took significant difference in positive lymph node (p<0.05).

Conclusions

Survivin takes an important part in regulating the expression of VEGF-C. VEGF-C could
influence the invasive ability mediated by survivin. The co-expression of survivin
and VEGF-C is more statistically significant to assess lymphatic metastasis in breast
cancer.

Virtual slides

Keywords:

Survivin; VEGF-C; Breast cancer; Lymphatic metastasis

Background

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer
death in females, accounting for 23% (1.38 million) of the total new cancer cases
and 14% (458,400) of the total cancer deaths in 2008 worldwide [1]. More than half of breast cancers are associated with axillary nodal involvement.
Metastasis and recurrence severely affect the quality and length of lives of breast
cancer patients. In the NCCN clinical practice guidelines of breast cancer, lymphatic
metastasis is considered the main criteria for clinical prognosis and target of systemic
treatment. Axillary lymph node status is the most important prognostic factor in breast
cancer, and prognosis declines with increasing number of tumor-positive lymph nodes
[2].

Lymphatic metastasis is one of the most important pathways of breast cancer systemic
metastasis, and is closely related to prognosis and therapy plans for breast cancer
patients. Many pathways have been suggested to be involved in the process of breast
cancer lymphatic metastasis. And some markers, such as EGFR [3] and BCRP [4], could be detected as the predictable target of breast cancer lymphatic metastasis.
Some researches have indicated that survivin and vascular endothelial growth factor-C
(VEGF-C) may take part in the course of lymphatic metastasis of breast cancer.

Survivin is one of the inhibitors of apoptosis protein (IAP). It regulates two important
cellular processes including inhibition of cell apoptosis and promoting cell proliferation
[5]. High levels of survivin mediate resistance of cancer cells to a series of anti-cancer
drugs [6], and it has been shown that up-regulation of survivin confers resistance of cancer
cells to radiotherapy [7,8]. Several researches in breast cancer, gastric cancer, oral squamous cell carcinoma
and colorectal cancer have shown that the expression of survivin is significantly
related to lymphatic metastasis, and that survivin is the prognostic marker for these
cancers [9-13]. However, how survivin controls lymphatic metastasis remains elusive.

VEGF-C, also called lymphatic vessel growth factor, is a lymphatic endothelial cell-stimulating
factor. VEGF-C affects blood vessel and lymphatic vessel through vascular endothelial
growth factor receptor-2 (VEGFR-2) and vascular endothelial growth factor receptor-3
(VEGFR-3), respectively, to improve tumor growth and metastasis [14]. High levels of VEGF-C have been detected inside or around tumors lymphatic vessels,
by which lymphatic and even distal metastasis are promoted [15]. High levels of VEGF-C also lead to lymphatic vessel formation in lymph nodes [16]. Many researches have also demonstrated that VEGF-C is involved in lymphatic invasion
in esophageal cancer, breast cancer, non-small cell lung cancer, and colorectal cancer
[17-20].

It has been suggested that both survivin and VEGF-C play important roles in tumor
lymphatic metastasis; however, studies on the relationship between the two are scarce.
In this study, we discuss the relationship between survivin and VEGF-C in breast cancer
and the pathway by which survivin may affect breast cancer lymphatic metastasis.

Material and methods

Clinical samples

A total of 108 breast cancer patients aged 32 to 63 with a mean age of 48 were involved
in this study. All patients were examined and monitored from 2009-2011 in the First
Affiliated Hospital of China Medical University in Shenyang, Liaoning province, China.
Breast cancer was diagnosed and classified into various stages according to the International
Union Against Cancer (UICC) and the TNM classification system published by the American
Joint Committee on Cancer (AJCC). Clinical information obtained from the records and
the histopathology reports included age, first diagnosis, tumor size and grade, oestrogen-receptor
(ER) and progesterone-receptor (PR) status, and lymph nodal involvement.

Cell culture

Human breast cancer cell line ZR-75-30 was preserved from Cell Resoure Center of Shanghai
Life Science Research Institute, Chinese Academy of Sciences. And it was cultured
in RPM1640 medium (Invitrogen Corporation, Carlsbad, California, USA) supplemented
with 50 μg/ml penicillin, 50 μg/ml streptomycin and 10% fetal bovine serum at 37°C
in a 5% CO2 environment. Long phase cells were collected after trypsin digestion by centrifugation
for 5 minute at 1,000 rpm, re-suspended in phosphate buffered saline (PBS), and counted
using a haemocytometer.

Construction and transfection of plasmid-ShRNA and lentivirus

Plasmids with survivin-shRNA and VEGF-C-shRNA were constructed by Shanghai GennePharma
Co., Ltd, and lentivirus with survivin gene was constructed by Shanghai GeneChem Co.,
Ltd. Plasmid-survivin-shRNA and plasmid-VEGF-C-shRNA, 10 μg shRNA and 25 μl Lipofectamine
2000 (Invitrogen Corporation, Carlsbad, California, USA) were mixed in 1350 μl 1640
medium without FBS and transfected into brest cancer cells. The mixture were added
into 25-cm2 culture flask that was previously plated with 1 × 106 ZR-75-30 breast cancer cells. Culture medium was replaced with complete 1640 medium
once six hours post inoculation and cells were collected after another 28 hours. Protein
and RNA were extracted for western blot and real-time PCR analysis, respectively.

Lentivirus with survivin gene was transfected into cells at an MOI of 20 and 0.75 μl
polyprene were added into each well of the 6-well plate containing 2 × 105 ZR-75-30 cells. Medium was replaced after 8 hours, and then cells were cultured at
37°C in a 5% CO2 environment.

RNA isolation and Real-time polymerase chain reaction

Total RNA was extracted using the guanidinium thiocyanate-phenol-chloroform method.
RNA yield and purity were determined photometrically (BioPhotometer, Eppendorf, Germany).
Reverse transcription was performed. Survivin and VEGF-C were amplified using real
time PCR. A total of 10 ng of reverse-transcribed total RNA was used as the template,
and PCR reaction contained 20 pmol/ml of each sense and antisense primer (Table 1) and SYBR Premix Ex Taq II (TaKaRa, Dalian, Shenyang, China) in a final volume of
20 μl. An ABI PRISM 7700 Sequence Detection System Instrument (Applied Biosystems)
was used for the amplification. Cycling conditions consisted of an initial denaturation
step at 95°C for 10 min as a ‘hot start’, followed by 40 cycles of 95°C for 15 s,
annealing temperature for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min.
GAPDH was used in each experiment as an endogenous control. Relative quantification
for a gene was expressed as fold changes over the control group. Fold changes were
calculated using the 2- ΔΔCt method.

Matrigel invasion assay

The migration capacity of breast cancer cells was determined in vitro using Transwell
Chambers (Corning, NY, USA) in which the two chambers were separated with matrigel
coated polycarbonate membrane (6.5-mm diameter inserts, 8 μm pore size). Breast cancer
cells (5 × 105/ml serum-free medium) were placed in the upper chamber (200 μl). The lower chamber
contained medium alone (500 μl). Chambers were assembled and kept in an incubator
for 24 hours. At the desired time point, cells from the upper surface of the membrane
were removed with gentle swabbing and the migrant cells on the lower surface of the
membrane were fixed by methanol and stained with crystal violet. Then membranes were
washed with PBS and mounted onto slide glasses. The membranes were visualized microscopically
(Olympus BX41) and cellular migration per sample was determined by counting the number
of stained cells in at least four to five randomly selected fields. Data are presented
as mean number of the migrating cells ± SD per microscopic field per sample. Each
cell migration experiment was repeated at least three times.

Immunohistochemical analysis

Immunohistochemistry was carried out on paraffin-embedded tumor specimens fixed in
4% buffered formalin. Four-micrometer-thick histological slides were de-paraffinized
in xylol and heated in 0.01 M citrate buffer for 25 min in a microwave oven. After
cooled for 20 min and washed in PBS, endogenous peroxidase was detected by incubating
samples with PBS containing 10% normal goat serum for 30 min. Then the sections were
incubated with each primary antibody overnight at 4°C. The primary antibodies were
anti-survivin (mouse monoclonal, Santa Cruz, CA., USA, 1:10) and anti-VEGF-C (rabbit
polyclonal, Abcam Inc, Cambridge, MA, UK, 1:200). A further wash in PBS was followed
by treatment with peroxidase-labeled polymer conjugated to goat anti-mouse or anti-rabbit
immunogloblins (Envison + kit; Dako, Glostrup, Denmark) as the secondary antibody
for 10 min at room temperature. The staining was visualized with diaminobenzidine
(DAB), followed by counterstaining with hematoxylin. For a negative control, PBS was
substituted for the primary antibody.

The degree of immunohistochemical staining was recorded on a scale of 0–3 according
to the percentages of staining and distributions within the cytoplasm. Tumors were
scored on a four-tier system: less than 10% of cancer cells staining was designated
negative (as degree 0), 10–20% positive staining was scored as degree 1+, 21–50% positive
staining was scored as degree 2+, and 51–100% positive staining was scored as degree
3+. We checked at least five 200 × visual fields for one glass slide under microscope,
counted the percentage of positive cells in every fields and got the mean percentage
of positive cells in a glass slide. At last, we got the score of the glass slide on
the base of percentage.

Statistical analysis

Data was expressed as the means of at least three different experiments ± SD. The
results were analyzed by chi-square test and Spearman analysis. p<0.05 was considered statistically significant.

Results

Transfection of plasmid and lentivirus

Plasmids with survivn shRNA and VEGF-C shRNA contained green fluorescent protein (GFP)
were transfected into cells using the method mentioned above. The transfection efficiency
was about 50%–70% after 24 hours.

Lentivirus-survivin with GFP was transfected into cells using the method mentioned
above. The transfection efficiency was nearly 100%.

Expressions of VEGF-C and survivin were positively correlated

In ZR-75-30 cells, when survivin was down-regulated, VEGF-C was down-regulated with
western blot method. When survivin was up-regulated, VEGF-C had a higher expression
level than that in normal cells. In ZR-75-30 cells over-expressing survivin, VEGF-C
expression level also decreased when survivin was down-regulated. Real-time PCR showed
that the level of VEGF-C mRNA positively correlated with the level of survivin mRNA
(Figure 1).

Survivin changed invasive ability of cells through VEGF-C

In the matrigel invasion assay, breast cancer cells with high levels of survivin were
more invasive than those with low levels of survivin, which suggested that survivin
played an important role in tumor cells migration. However, down-regulated VEGF-C
in these cells significantly reduced the number of breast cancer cells that could
migrate through the polycarbonate membrane (Figure 2).

Figure 2.Matrigel invasion assay from different processed cells.a,b,c and d show the invasive cells of group 30, group 30 + lv, group 30 + lv-S and group 30 + lv-S + p-V
under a microscope(200×). e shows the mean number counted from above visual field. * means statistically significant
different between 30 + lv-S and ZR-75-30 cells. ** means significant different between
30 + lv-S and 30 + lv-S + p-V(both p<0.05).

Survivin and VEGF-C expression in tumor tissue and lymph node

Survivin and VEGF-C mainly localized in the cytoplasm, but could also be detected
in the nuclei with nuclear-specific dye. Survivin was expressed in the breast cancer
tissue of 83.3% of the patients, among which degree 1-3+ were expressed at 26.7%,
50.0%, 23.3%. In the same group of patients, VEGF-C was expressed at 77.8% and degree
1-3+ were 28.6%, 53.6%, 17.9%. In the lymph node tissue, both survivin and VEGF-C
were expressed at higher levels in positive LN than in negative LN (Figure 3).

Figure 3.Results of immunohistochemical staining of breast cancer tissue and lymph node.a shows survivin expression in the order of degree 0-3+ in breast cancer tissue; b shows VEGF-C expression in the order of degree 0-3+ in breast cancer tissue; c shows survivin and VEGF-C expressions in negative and metastatic lymph node respectively(200×).

Pathological analysis of patients

Survivin and VEGF-C were expressed at higher levels in patients with lymphatic metastasis,
and stage III, IV breast cancers (Table 2). The expression levels were also statistically different in tumors of different
sizes. However, there were no differences in the expression of the two genes in patients
with different age, histological grade, pathological style, or ER/PR status. The results
showed that higher expression was detected in C-erBb-2 positive but not in C-erBb-2
negative patients.

Table 2.Association of survivin and VEGF-C protein expression with clinicopathological features
in breast cancer patients

There was significant difference between the levels of VEGF-C and survivin expression
(p<0.05) (Table 3). When the level of survivin expression increased from degree 0 to 3, the level of
VEGF-C expression increased correspondingly. And the Pearson coefficient of contingency
C = 0.514, which means an actual expression relationship between VEGF-C and survivin.

Table 3.Relationship of expression extent between surivivin and VEGF-C

In the patients with co-expression of survivin and VEGF-C, the lymph node positive
rate is higher than the patients with the single or none expression of survivin and
VEGF-C (p<0.05) (Table 4). This result indicated that survivin co-operated with VEGF-C in lymphatic metastasis.

Table 4.Relationship of co-expression of survivin and VEGF-C with lymph node involvement

Discussion

The expression of survivin is high during fetal development but low in healthy adult
tissues. However, in most malignant tumors, the expression of survivin increases.
As a result, survivin has been considered a potential tumor marker and an important
therapeutic target [5]. Studies show that the expression of survivin in many kinds of tumors correlates
with lymphatic metastasis. Our results suggest that survivin may influence breast
cancer lymphatic metastasis and distal invasion through VEGF-C.

Researches on different tumors have indicated that the up-regulation of VEGF-C promotes
tumor lymphatic vessel formation and increases lymph node metastasis. VEGF-C could
form lots of lymphatic vessel inside or around the tumor, by which promoting tumor
metastasis to lymph node and even distal organs [15]. Then it could make a good condition for advanced metastasis and diffusion.

We showed that the protein and mRNA expression of VEGF-C are controlled by survivin.
So, there must be a notal point by which the expression of VEGF-C could be regulated
by survivin. Cox-2 activation is highly correlated with VEGF-C expression [21], and through its downstream molecules, cox-2 is able to up-regulate the expression
of VEGF-C in cancer cells [22,23]. As an important regulator of apoptosis, cox-2 is usually over-expressed with survivin
in hepatocellular carcinoma, surperficial urothelial carcinoma and endometrial carcinoma
[24-26]. It is possible that cox-2 may be the notal point to link survivin and VEGF-C expressions.

It has been suggested that the interaction of XIAP and survivin promotes the invasion
of tumor cells and enhances the metastatic spread in vivo [27]. Khan et al. have shown that synthetic survivin enhances the proliferation, drug
resistance, and cellular invasion of tumor cells [28]. It has been suggested that VEGF-C could promote the VEGFR3 positive tumor cells
invade to lymphatic vessels through autocrine and CCR-7 dependent paracrine mechanism
[29]. VEGF-C was also proven to control tumor cells growth and invasion by atuocrine mechanism
in the carcinoma of gallbladder [30]. In accordance with this result, our results show that the invasiveness of breast
cancer cell increases when survivin is over-expressed, and significantly decreases
when survivn is over-expressed while VEGF-C is down-regulated. So VEGF-C may play
an important role in enhancing the invasiveness in tumor cells caused by survivin.
On the other hand, in positive lymph node, survivin and VEGF-C both express at high
levels, which may indicate that both of them play important roles in lymphatic metastasis
and invasion in breast cancer.

Several researches found that survivin and VEGF-C took high level expression in the
breast cancer, also had positive relationship with positive lymphatic metastasis respectively
[9,31,32]. And not only in the primary breast tumor, survivin was also found high level expression
in circulating tumor cells in peripheral blood through RT-PCR ELISA method [33]. It suggested that the tumor cells with survivin high expression showed great invasive
and metastatic ability. In our study, it has been proven that survivin and VEGF-C
both are closely related with lymphatic metastasis in different tumors. We show in
our study that survivin and VEGF-C expression are positively correlated, and the co-expression
of the two is also positively correlated with positive lymph node. This result supports
the conclusion based on previous finding that changes in survivin expression induce
the changes in VEGF-C expression.

Conclusion

As a conclusion, survivin is related with breast cancer lymphatic metastasis. It gets
correlation with the rate of lymph node metastasis and the invasive ability of breast
cancer cells. VEGF-C may play an important role in these processes. Survivin can influenced
the expression of VEGF-C to reduce breast cancer lymphatic metastasis and invasion,
which helps to decrease death risk of breast cancer. Meanwhile, survivin and VEGF-C
can be used simultaneously as important markers to access lymphatic metastasis and
distal invasion of breast cancer.

Competing interests

All authors declare no competing interests.

Authors’ contributions

XC and SM carried out the design and western blot of this research, MG and CZ participated
in real-time PCR and immunohistochemistry of this research, WQ performed the Statistical
Analysis, XZ was the director of this research and helped to draft the manuscript.
All authors read and approved the final manuscript.

Acknowledgements

This work was partially supported by grants from the Scientific Research Foundation
for Returned Scholars of Ministry of Education of China (2008), Hi-Tech Research Development
Program of China (863 Program, 2006AA02Z493) and the National Natural Science Foundation
of China (No. 81071900 and 81172199).